RNA interference mediated inhibition of muscarinic colinergic receptor gene expression using short interfering nucleic acid (siNA)

ABSTRACT

The present invention relates to compounds, compositions, and methods useful for modulating the expression of genes associated with respiratory and pulmonary disease, such as cholinergic muscarinic receptor genes, using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of cholinergic muscarinic receptor genes, or other genes involved in pathways of cholinergic muscarinic receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of M3 muscarinic acetylcholine receptor or cholinergic receptor muscarinic 3 (CHRM3).

This application is a continuation of U.S. patent application Ser. No.12/203,731, filed on Sep. 3, 2009, which is a continuation of U.S.patent application Ser. No. 10/798,090, filed on Mar. 11, 2004, which isa continuation-in-part of U.S. patent application Ser. No. 10/757,803,filed Jan. 14, 2004, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/720,448, filed Nov. 24, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/693,059,filed Oct. 23, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/444,853, filed May 23, 2003, which is acontinuation-in-part of International Patent Application No.PCT/US03/05346, filed Feb. 20, 2003, and a continuation-in-part ofInternational Patent Application No. PCT/US03/05028, filed Feb. 20,2003, both of which claim the benefit of U.S. Provisional ApplicationNo. 60/358,580, filed Feb. 20, 2002, U.S. Provisional Application No.60/363,124, filed Mar. 11, 2002, U.S. Provisional Application No.60/386,782, filed Jun. 6, 2002, U.S. Provisional Application No.60/406,784, filed Aug. 29, 2002, U.S. Provisional Application No.60/408,378, filed Sep. 5, 2002, U.S. Provisional Application No.60/409,293, filed Sep. 9, 2002, and U.S. Provisional Application No.60/440,129, filed Jan. 15, 2003, and parent U.S. patent application Ser.No. 10/798,090 is also a continuation-in-part of U.S. patent applicationSer. No. 10/427,160, filed Apr. 30, 2003 and International PatentApplication No. PCT/US02/15876, filed May 17, 2002. The instantapplication claims the benefit of all the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing57USCNT2,”created on Dec. 8, 2009, which is 102,591 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compounds, compositions, and methods forthe study, diagnosis, and treatment of diseases and conditions thatrespond to the modulation of cholinergic muscarinic receptor geneexpression and/or activity. The present invention also concernscompounds, compositions, and methods relating to diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in cholinergic muscarinic receptor gene expression pathways orother cellular processes that mediate the maintenance or development ofsuch diseases and conditions. Specifically, the invention relates tosmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable ofmediating RNA interference (RNAi) against cholinergic muscarinicreceptor gene expression.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double-strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy(2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of an siRNAduplex is required for siRNA activity and that ATP is utilized tomaintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al. International PCT Publication No. WO 02/38805, describecertain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certainsingle-stranded siRNA constructs, including certain 5′-phosphorylatedsingle-stranded siRNAs that mediate RNA interference in HeLa cells.Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13,83-105, describe certain chemically and structurally modified siRNAmolecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certainchemically and structurally modified siRNA molecules.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating the expression of genes associated with respiratory andpulmonary disease, such as cholinergic muscarinic receptor genes, usingshort interfering nucleic acid (siNA) molecules. This invention alsorelates to compounds, compositions, and methods useful for modulatingthe expression and activity of cholinergic muscarinic receptor genes, orother genes involved in pathways of cholinergic muscarinic receptor geneexpression and/or activity by RNA interference (RNAi) using smallnucleic acid molecules. In particular, the instant invention featuressmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methodsused to modulate the expression of M3 muscarinic acetylcholine receptor(CHRM3).

An siNA of the invention can be unmodified or chemically modified. AnsiNA of the instant invention can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. The instant invention alsofeatures various chemically modified synthetic short interfering nucleicacid (siNA) molecules capable of modulating repeat expansion geneexpression or activity in cells by RNA interference (RNAi). The use ofchemically modified siNA improves various properties of native siNAmolecules through increased resistance to nuclease degradation in vivoand/or through improved cellular uptake. Further, contrary to earlierpublished studies, siNA having multiple chemical modifications retainsits RNAi activity. The siNA molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, diagnostic,target validation, genomic discovery, genetic engineering, andpharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofcholinergic muscarinic receptor genes encoding proteins, such asproteins comprising cholinergic muscarinic receptors and ligandsthereof, associated with the maintenance and/or development ofrespiratory and/or pulmonary diseases, such as genes encoding sequencescomprising those sequences referred to by GenBank Accession Nos. shownin Table I, referred to herein generally as CHRM3. The description belowof the various aspects and embodiments of the invention is provided withreference to exemplary M3 muscarinic acetylcholine receptor referred toherein as CHRM3. However, the various aspects and embodiments are alsodirected to other muscarinic acetylcholine receptor genes, such asCHRM1, CHRM2, CHRM4, CHRM5 and polymorphisms (e.g., SNPs) associatedwith such muscarinic acetylcholine receptor genes (including CHRM3polymorphisms, see for example Choi et al., WO 01/029176 and Hall, 2002,Respir. Res., 3, 10). As such, the various aspects and embodiments arealso directed to other genes that are involved in CHRM3 mediatedpathways of signal transduction or gene expression that are involved inthe progression, development, and/or maintenance of disease (e.g.,chronic obstructive pulmonary disease or “COPD”, asthma, allergicrhinitis, sinusitis, pulmonary vasoconstriction, inflammation,allergies, impeded respiration, respiratory distress syndrome, cysticfibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema,irritable bowel syndrome, Alzheimer's disease, urinary incontinence),including genes encoding CHRM3 ligands and agonists. These additionalgenes can be analyzed for target sites using the methods described forCHRM3 genes herein. Thus, the modulation of other genes and the effectsof such modulation of the other genes can be performed, determined, andmeasured as described herein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a cholinergic muscarinic receptor (e.g., CHRM3) gene, wherein saidsiNA molecule comprises about 19 to about 21 base pairs.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a CHRM3 gene, for example, wherein theCHRM3 gene comprises CHRM3 encoding sequence. In one embodiment, theinvention features an siNA molecule that down-regulates expression of aCHRM3 gene, for example, wherein the CHRM3 gene comprises CHRM3non-coding sequence or regulatory elements involved in CHRM3 geneexpression.

In one embodiment, the invention features an siNA molecule having RNAiactivity against CHRM3 RNA, wherein the siNA molecule comprises asequence complementary to any RNA having CHRM3 encoding sequence, suchas those sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features an siNA molecule having RNAiactivity against CHRM3 RNA, wherein the siNA molecule comprises asequence complementary to an RNA having other CHRM3 encoding sequence,for example other mutant CHRM3 genes not shown in Table I but known inthe art to be associated with respiratory and/or pulmonary disease(e.g., chronic obstructive pulmonary disease or “COPD”, asthma, allergicrhinitis, sinusitis, pulmonary vasoconstriction, inflammation,allergies, impeded respiration, respiratory distress syndrome, cysticfibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema,irritable bowel syndrome, Alzheimer's disease, urinary incontinence)Chemical modifications as shown in Tables III and IV or otherwisedescribed herein can be applied to any siNA construct of the invention.In another embodiment, an siNA molecule of the invention includesnucleotide sequence that can interact with nucleotide sequence of aCHRM3 gene and thereby mediate silencing of CHRM3 gene expression, forexample, wherein the siNA mediates regulation of CHRM3 gene expressionby cellular processes that modulate the chromatin structure of the CHRM3gene and prevent transcription of the CHRM3 gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of CHRM3 proteins arising from CHRM3haplotype polymorphisms that are associated with disease, (e.g.,associated with a gain of function). Analysis of CHRM3 genes, or CHRM3protein or RNA levels can be used to identify subjects with suchpolymorphisms or those subjects who are at risk of developing diseasesdescribed herein. These subjects are amenable to treatment, for example,treatment with siNA molecules of the invention and any other compositionuseful in treating diseases related to CHRM3 gene expression. As such,analysis of CHRM3 protein or RNA levels can be used to determinetreatment type and the course of therapy in treating a subject.Monitoring of CHRM3 protein or RNA levels can be used to predicttreatment outcome and to determine the efficacy of compounds andcompositions that modulate the level and/or activity of certain CHRM3proteins associated with disease.

In another embodiment, the invention features an siNA moleculecomprising nucleotide sequence, for example, nucleotide sequence in theantisense region of the siNA molecule that is complementary to anucleotide sequence or portion of sequence of a CHRM3 gene. In anotherembodiment, the invention features an siNA molecule comprising a region,for example, the antisense region of the siNA construct, complementaryto a sequence comprising a CHRM3 gene sequence or a portion thereof.

In one embodiment, the antisense region of CHRM3 siNA constructs cancomprise a sequence complementary to sequence having any of SEQ ID NOs.1-99 and 199-206. In one embodiment, the antisense region can alsocomprise sequence having any of SEQ ID NOs. 100-198, 215-222, 231-238,247-254, 263-270, 279-286, 297, 299, 301, or 304. In another embodiment,the sense region of the CHRM3 constructs can comprise sequence havingany of SEQ ID NOs. 1-99, 199-214, 223-230, 239-246, 255-262, 271-278,296, 298, 300, 302, or 303. The sense region can comprise a sequence ofSEQ ID NO. 287 and the antisense region can comprise a sequence of SEQID NO. 288. The sense region can comprise a sequence of SEQ ID NO. 289and the antisense region can comprise a sequence of SEQ ID NO. 290. Thesense region can comprise a sequence of SEQ ID NO. 291 and the antisenseregion can comprise a sequence of SEQ ID NO. 292. The sense region cancomprise a sequence of SEQ ID NO. 293 and the antisense region cancomprise a sequence of SEQ ID NO. 290. The sense region can comprise asequence of SEQ ID NO. 294 and the antisense region can comprise asequence of SEQ ID NO. 290. The sense region can comprise a sequence ofSEQ ID NO. 293 and the antisense region can comprise a sequence of SEQID NO. 295.

In one embodiment, an siNA molecule of the invention comprises any ofSEQ ID NOs. 1-304. The sequences shown in SEQ ID NOs: 1-304 are notlimiting. An siNA molecule of the invention can comprise any contiguousCHRM3 sequence (e.g., about 19 to about 25, or about 19, 20, 21, 22, 23,24 or 25 contiguous CHRM3 nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 19 to about 29 (e.g., about 19, 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein the antisensestrand is complementary to a RNA sequence encoding a CHRM3 protein, andwherein said siNA further comprises a sense strand having about 19 toabout 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences with at least about 19 complementarynucleotides.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, wherein theantisense region is complementary to a RNA sequence encoding a CHRM3protein, and wherein said siNA further comprises a sense region havingabout 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or more) nucleotides, wherein said sense region and saidantisense region comprise a linear molecule with at least about 19complementary nucleotides.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a CHRM3 protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a CHRM3 gene or a portion thereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a CHRM3 protein or a portion thereof. The siNAmolecule further comprises a sense region, wherein said sense regioncomprises a nucleotide sequence of a CHRM3 gene or a portion thereof.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a CHRM3 gene. Because CHRM3genes can share some degree of sequence homology with each other, siNAmolecules can be designed to target a class of CHRM3 genes oralternately specific CHRM3 genes (e.g., polymorphic variants) byselecting sequences that are either shared amongst different CHRM3targets or alternatively that are unique for a specific CHRM3 target.Therefore, in one embodiment, the siNA molecule can be designed totarget conserved regions of CHRM3 RNA sequence having homology betweenseveral CHRM3 gene variants so as to target a class of CHRM3 genes withone siNA molecule. Accordingly, in one embodiment, the siNA molecule ofthe invention modulates the expression of one or both CHRM3 alleles in asubject. In another embodiment, the siNA molecule can be designed totarget a sequence that is unique to a specific CHRM3 RNA sequence (e.g.,a single CHRM3 allele or CHRM3 SNP) due to the high degree ofspecificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplexes containing about 19 basepairs between oligonucleotides comprising about 19 to about 25 (e.g.,about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplexes withoverhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3)nucleotides, for example, about 21-nucleotide duplexes with about 19base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs.

In one embodiment, the invention features one or more chemicallymodified siNA constructs having specificity for CHRM3 expressing nucleicacid molecules, such as RNA encoding a CHRM3 protein. Non-limitingexamples of such chemical modifications include without limitationphosphorothioate internucleotide linkages, 2′-deoxyribonucleotides,2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, and terminal glyceryl and/or inverted deoxy abasic residueincorporation. These chemical modifications, when used in various siNAconstructs, are shown to preserve RNAi activity in cells while at thesame time, dramatically increasing the serum stability of thesecompounds. Furthermore, contrary to the data published by Parrish etal., supra, applicant demonstrates that multiple (greater than one)phosphorothioate substitutions are well-tolerated and confer substantialincreases in serum stability for modified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100% modified nucleotides). The actual percentage ofmodified nucleotides present in a given siNA molecule will depend on thetotal number of nucleotides present in the siNA. If the siNA molecule issingle-stranded, the percent modification can be based upon the totalnumber of nucleotides present in the single-stranded siNA molecules.Likewise, if the siNA molecule is double-stranded, the percentmodification can be based upon the total number of nucleotides presentin the sense strand, antisense strand, or both the sense and antisensestrands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a CHRM3gene. In one embodiment, a double-stranded siNA molecule comprises oneor more chemical modifications and each strand of the double-strandedsiNA is about 21 nucleotides long. In one embodiment, thedouble-stranded siNA molecule does not contain any ribonucleotides. Inanother embodiment, the double-stranded siNA molecule comprises one ormore ribonucleotides. In one embodiment, each strand of thedouble-stranded siNA molecule comprises about 19 to about 23 (e.g.,about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides,wherein each strand comprises about 19 nucleotides that arecomplementary to the nucleotides of the other strand. In one embodiment,one of the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence or aportion thereof of the CHRM3 gene, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the CHRM3 gene or aportion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene comprising an antisense region, wherein the antisenseregion comprises a nucleotide sequence that is complementary to anucleotide sequence of the CHRM3 gene or a portion thereof, and a senseregion, wherein the sense region comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the CHRM3 gene or aportion thereof. In one embodiment, the antisense region and the senseregion each comprise about 19 to about 23 (e.g. about 19, 20, 21, 22, or23) nucleotides, wherein the antisense region comprises about 19nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the CHRM3 geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, an siNA molecule of the invention comprising modificationsdescribed herein (e.g., comprising nucleotides having Formulae I-VII orsiNA constructs comprising Stab00-Stab22 or any combination thereof (seeTable IV)) and/or any length described herein can comprise blunt ends orends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In a non-limiting example, a blunt ended siNA molecule hasa number of base pairs equal to the number of nucleotides present ineach strand of the siNA molecule. In another example, an siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, an siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, an siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 18 to about 30nucleotides (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides). Other nucleotides present in a blunt ended siNAmolecule can comprise mismatches, bulges, loops, or wobble base pairs,for example, to modulate the activity of the siNA molecule to mediateRNA interference.

By “blunt ends” is meant symmetric termini or termini of adouble-stranded siNA molecule having no overhanging nucleotides. The twostrands of a double-stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. The sense region can be connected to the antisenseregion via a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene, wherein the siNA molecule comprises about 19 to about21 base pairs, and wherein each strand of the siNA molecule comprisesone or more chemical modifications. In another embodiment, one of thestrands of the double-stranded siNA molecule comprises a nucleotidesequence that is complementary to a nucleotide sequence of a CHRM3 geneor a portion thereof, and the second strand of the double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence or a portion thereof of the CHRM3 gene. In anotherembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence of a CHRM3 gene or a portion thereof, and the second strand ofthe double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or a portion thereof ofthe CHRM3 gene. In another embodiment, each strand of the siNA moleculecomprises about 19 to about 23 nucleotides, and each strand comprises atleast about 19 nucleotides that are complementary to the nucleotides ofthe other strand. The CHRM3 gene can comprise, for example, sequencesreferred to Table I.

In one embodiment, an siNA molecule of the invention comprises noribonucleotides. In another embodiment, an siNA molecule of theinvention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a CHRM3 gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the CHRM3 gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 19 to about 23 nucleotides and theantisense region comprises at least about 19 nucleotides that arecomplementary to nucleotides of the sense region. The CHRM3 gene cancomprise, for example, sequences referred to Table I.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a CHRM3 gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In another embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The CHRM3 genecan comprise, for example, sequences referred to Table I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the CHRM3 geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thesiNA molecule has one or more modified pyrimidine and/or purinenucleotides. In one embodiment, the pyrimidine nucleotides in the senseregion are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In one embodiment, thepyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in theantisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. Inanother embodiment of any of the above-described siNA molecules, anynucleotides present in a non-complementary region of the sense strand(e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule, and wherein the fragment comprising the sense regionincludes a terminal cap moiety at the 5′-end, the 3′-end, or both of the5′ and 3′ ends of the fragment. In another embodiment, the terminal capmoiety is an inverted deoxy abasic moiety or glyceryl moiety. In anotherembodiment, each of the two fragments of the siNA molecule compriseabout 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, of lengthbetween about 12 and about 36 nucleotides. In another embodiment, allpyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides. In another embodiment, the modified nucleotidesin the siNA include at least one 2′-deoxy-2′-fluoro cytidine or2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, themodified nucleotides in the siNA include at least one 2′-deoxy-2′-fluorocytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. Inanother embodiment, all uridine nucleotides present in the siNA are2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, allcytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In another embodiment, all adenosine nucleotides present inthe siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In anotherembodiment, all guanosine nucleotides present in the siNA are2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further compriseat least one modified internucleotidic linkage, such as phosphorothioatelinkage. In another embodiment, the 2′-deoxy-2′-fluoronucleotides arepresent at specifically selected locations in the siNA that aresensitive to cleavage by ribonucleases, such as locations havingpyrimidine nucleotides.

In one embodiment, the invention features a method of increasing thestability of an siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In another embodiment, all pyrimidine nucleotides present inthe siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In anotherembodiment, the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-deoxy-2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, alluridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In another embodiment, all cytidine nucleotides present inthe siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In anotherembodiment, all adenosine nucleotides present in the siNA are2′-deoxy-2′-fluoro adenosine nucleotides. In another embodiment, allguanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inanother embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the CHRM3 geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thepurine nucleotides present in the antisense region comprise2′-deoxy-purine nucleotides. In an alternative embodiment, the purinenucleotides present in the antisense region comprise 2′-O-methyl purinenucleotides. In either of the above embodiments, the antisense regioncan comprise a phosphorothioate internucleotide linkage at the 3′ end ofthe antisense region. Alternatively, in either of the above embodiments,the antisense region can comprise a glyceryl modification at the 3′ endof the antisense region. In another embodiment of any of theabove-described siNA molecules, any nucleotides present in anon-complementary region of the antisense strand (e.g. overhang region)are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of theinvention comprises sequence complementary to a portion of a CHRM3transcript having sequence unique to a particular CHRM3 disease relatedallele, such as sequence comprising a SNP associated with the diseasespecific allele. As such, the antisense region of an siNA molecule ofthe invention can comprise sequence complementary to sequences that areunique to a particular allele to provide specificity in mediatingselective RNAi against the disease related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a CHRM3 gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment about 19 nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule and wherein atleast two 3′ terminal nucleotides of each fragment of the siNA moleculeare not base-paired to the nucleotides of the other fragment of the siNAmolecule. In one embodiment, each of the two 3′ terminal nucleotides ofeach fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide,such as a 2′-deoxy-thymidine. In another embodiment, all 21 nucleotidesof each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule. Inanother embodiment, about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the CHRM3 gene. In another embodiment, about 21 nucleotidesof the antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the CHRM3 gene. In any of theabove embodiments, the 5′-end of the fragment comprising said antisenseregion can optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa CHRM3 RNA sequence (e.g., wherein said target RNA sequence is encodedby a CHRM3 gene involved in the CHRM3 pathway), wherein the siNAmolecule does not contain any ribonucleotides and wherein each strand ofthe double-stranded siNA molecule is about 21 nucleotides long. Examplesof non-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table IV in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab 18/20.

In one embodiment, the invention features a chemically synthesizeddouble-stranded RNA molecule that directs cleavage of a CHRM3 RNA viaRNA interference, wherein each strand of said RNA molecule is about 21to about 23 nucleotides in length; one strand of the RNA moleculecomprises nucleotide sequence having sufficient complementarity to theCHRM3 RNA for the RNA molecule to direct cleavage of the CHRM3 RNA viaRNA interference; and wherein at least one strand of the RNA moleculecomprises one or more chemically modified nucleotides described herein,such as deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to down-regulateexpression of a CHRM3 gene, wherein the siNA molecule comprises one ormore chemical modifications and each strand of the double-stranded siNAis about 18 to about 28 or more (e.g., 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28 or more) nucleotides long.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits expressionof a CHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aCHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aCHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA that encodes aprotein or portion thereof, the other strand is a sense strand whichcomprises nucleotide sequence that is complementary to a nucleotidesequence of the antisense strand and wherein a majority of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, the inventionfeatures a double-stranded short interfering nucleic acid (siNA)molecule that inhibits expression of a CHRM3 gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of CHRM3 RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, each strand of thesiNA molecule comprises about 18 to about 29 or more (e.g., about 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more) nucleotides, whereineach strand comprises at least about 18 nucleotides that arecomplementary to the nucleotides of the other strand. In anotherembodiment, the siNA molecule is assembled from two oligonucleotidefragments, wherein one fragment comprises the nucleotide sequence of theantisense strand of the siNA molecule and a second fragment comprisesnucleotide sequence of the sense region of the siNA molecule. In yetanother embodiment, the sense strand is connected to the antisensestrand via a linker molecule, such as a polynucleotide linker or anon-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aCHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification, and wherein each of the two strands of the siNAmolecule comprises about 21 nucleotides. In one embodiment, about 21nucleotides of each strand of the siNA molecule are base-paired to thecomplementary nucleotides of the other strand of the siNA molecule. Inanother embodiment, about 19 nucleotides of each strand of the siNAmolecule are base-paired to the complementary nucleotides of the otherstrand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In another embodiment, each strand of the siNA molecule is base-pairedto the complementary nucleotides of the other strand of the siNAmolecule. In another embodiment, about 19 nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the CHRM3 RNA or aportion thereof. In another embodiment, about 21 nucleotides of theantisense strand are base-paired to the nucleotide sequence of the CHRM3RNA or a portion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aCHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the 5′-end of the antisense strand optionally includes aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aCHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the CHRM3 RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aCHRM3 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of CHRM3 RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the CHRM3 RNA or aportion thereof that is present in the CHRM3 RNA.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding CHRM3 and thesense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against CHRM3 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotidescomprising a backbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring or chemicallymodified, each X and Y is independently O, S, N, alkyl, or substitutedalkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Zare optionally not all O. In another embodiment, a backbone modificationof the invention comprises a phosphonoacetate and/orthiophosphonoacetate internucleotide linkage (see for example Sheehan etal., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against CHRM3 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula II at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotides or non-nucleotides ofFormula II at the 5′-end of the sense strand, the antisense strand, orboth strands. In anther non-limiting example, an exemplary siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically modified nucleotides or non-nucleotidesof Formula II at the 3′-end of the sense strand, the antisense strand,or both strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against CHRM3 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, napthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula III at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotide(s) or non-nucleotide(s) ofFormula III at the 5′-end of the sense strand, the antisense strand, orboth strands. In anther non-limiting example, an exemplary siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide ofFormula III at the 3′-end of the sense strand, the antisense strand, orboth strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against CHRM3 inside a cell or reconstituted invitro system, wherein the chemical modification comprises a 5′-terminalphosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against CHRM3 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or morephosphorothioate internucleotide linkages. For example, in anon-limiting example, the invention features a chemically modified shortinterfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 ormore phosphorothioate internucleotide linkages in one siNA strand. Inyet another embodiment, the invention features a chemically modifiedshort interfering nucleic acid (siNA) individually having about 1, 2, 3,4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in bothsiNA strands. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siNA duplex, forexample in the sense strand, the antisense strand, or both strands. ThesiNA molecules of the invention can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the sense strand, the antisense strand,or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, or both strands. In yet anothernon-limiting example, an exemplary siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5,specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically modified, wherein each strand is about 18 to about 27(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides inlength, wherein the duplex has about 18 to about 23 (e.g., about 18, 19,20, 21, 22, or 23) base pairs, and wherein the chemical modificationcomprises a structure having any of Formulae I-VII. For example, anexemplary chemically modified siNA molecule of the invention comprises aduplex having two strands, one or both of which can be chemicallymodified with a chemical modification having any of Formulae I-VII orany combination thereof, wherein each strand consists of about 21nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang,and wherein the duplex has about 19 base pairs. In another embodiment,an siNA molecule of the invention comprises a single-stranded hairpinstructure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40,45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 toabout 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and whereinthe siNA can include a chemical modification comprising a structurehaving any of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprises alinear oligonucleotide having about 42 to about 50 (e.g., about 42, 43,44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modifiedwith a chemical modification having any of Formulae I-VII or anycombination thereof, wherein the linear oligonucleotide forms a hairpinstructure having about 19 base pairs and a 2-nucleotide 3′-terminalnucleotide overhang. In another embodiment, a linear hairpin siNAmolecule of the invention contains a stem loop motif, wherein the loopportion of the siNA molecule is biodegradable. For example, a linearhairpin siNA molecule of the invention is designed such that degradationof the loop portion of the siNA molecule in vivo can generate adouble-stranded siNA molecule with 3′-terminal overhangs, such as3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 23(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group thatcan be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In another embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprises alinear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemicallymodified with one or more chemical modifications having any of FormulaeI-VII or any combination thereof, wherein the linear oligonucleotideforms an asymmetric hairpin structure having about 3 to about 18 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) basepairs and a 5′-terminal phosphate group that can be chemically modifiedas described herein (for example a 5′-terminal phosphate group havingFormula IV). In another embodiment, an asymmetric hairpin siNA moleculeof the invention contains a stem loop motif, wherein the loop portion ofthe siNA molecule is biodegradable. In another embodiment, an asymmetrichairpin siNA molecule of the invention comprises a loop portioncomprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides in length, wherein the sense region is about3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18) nucleotides in length, wherein the sense region and theantisense region have at least 3 complementary nucleotides, and whereinthe siNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises an asymmetric double-stranded structure having separatepolynucleotide strands comprising sense and antisense regions, whereinthe antisense region is about 18 to about 22 (e.g., about 18, 19, 20,21, or 22) nucleotides in length and wherein the sense region is about 3to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double-stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) basepairs, and wherein the siNA can include a chemical modification, whichcomprises a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically modified siNA molecule ofthe invention comprises a circular oligonucleotide having about 42 toabout 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotidesthat is chemically modified with a chemical modification having any ofFormulae I-VII or any combination thereof, wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R1, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R1, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3,R8 or R13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of an siNA molecule of the invention. For example, a moietyhaving Formula V, VI or VII can be present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense strand, the sense strand, orboth antisense and sense strands of the siNA molecule. In addition, amoiety having Formula VII can be present at the 3′-end or the 5′-end ofa hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against CHRM3 inside a cell orreconstituted in vitro system comprising a sense region, wherein one ormore pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of these chemicallymodified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.In any of these described embodiments, the purine nucleotides present inthe sense region are alternatively 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides) and one or more purine nucleotides present in the antisenseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Also, in any of these embodiments, one or more purine nucleotidespresent in the sense region are alternatively purine ribonucleotides(e.g., wherein all purine nucleotides are purine ribonucleotides oralternately a plurality of purine nucleotides are purineribonucleotides) and any purine nucleotides present in the antisenseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a Northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double-stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against CHRM3 inside a cell or reconstituted invitro system, wherein the chemical modification comprises a conjugatecovalently attached to the chemically modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002,incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemisty 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides not having anyribonucleotides (e.g., nucleotides having a 2′-OH group) present in theoligonucleotides. In another example, an siNA molecule can be assembledfrom a single oligonucleotide where the sense and antisense regions ofthe siNA are linked or circularized by a nucleotide or non-nucleotidelinker as described herein, wherein the oligonucleotide does not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotide. Applicant has surprisingly found that the presenceof ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) withinthe siNA molecule is not required or essential to support RNAi activity.As such, in one embodiment, all positions within the siNA can includechemically modified nucleotides and/or non-nucleotides such asnucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI,or VII or any combination thereof to the extent that the ability of thesiNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single-stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single-stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single-stranded siNAmolecule of the invention comprises about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In yetanother embodiment, the single-stranded siNA molecule of the inventioncomprises one or more chemically modified nucleotides or non-nucleotidesdescribed herein. For example, all the positions within the siNAmolecule can include chemically modified nucleotides such as nucleotideshaving any of Formulae I-VII, or any combination thereof to the extentthat the ability of the siNA molecule to support RNAi activity in a cellis maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence,wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 10, that is optionally present at the 3′-end and/or the5′-end. The siNA optionally further comprises about 1 to about 4 or more(e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the3′-end of the siNA molecule, wherein the terminal nucleotides canfurther comprise one or more (e.g., 1, 2, 3, 4 or more)phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages, and wherein the siNA optionally furthercomprises a terminal phosphate group, such as a 5′-terminal phosphategroup. In any of these embodiments, any purine nucleotides present inthe antisense region are alternatively 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA (i.e., purine nucleotides present in the senseand/or antisense region) can alternatively be locked nucleic acid (LNA)nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides oralternately a plurality of purine nucleotides are LNA nucleotides).Also, in any of these embodiments, any purine nucleotides present in thesiNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-methoxyethyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides). In another embodiment, any modified nucleotides present inthe single-stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically modified nucleotides present in the single-strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, the invention features a method for modulating theexpression of a CHRM3 gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the CHRM3 gene; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the CHRM3 genein the cell.

In one embodiment, the invention features a method for modulating theexpression of a CHRM3 gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the CHRM3 gene and wherein the sense strand sequence of the siNAcomprises a sequence identical or substantially similar to the sequenceof the target RNA; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the CHRM3 genein the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one CHRM3 gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the CHRM3 genes; and (b) introducing the siNAmolecules into a cell under conditions suitable to modulate theexpression of the CHRM3 genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more CHRM3 genes within a cell comprising: (a)synthesizing one or more siNA molecules of the invention, which can bechemically modified, wherein the siNA strands comprise sequencescomplementary to RNA of the CHRM3 genes and wherein the sense strandsequences of the siNAs comprise sequences identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the CHRM3 genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one CHRM3 gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the CHRM3 gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecule into a cell under conditions suitable to modulate theexpression of the CHRM3 genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a CHRM3 genein a tissue explant comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the CHRM3 gene; and(b) introducing the siNA molecule into a cell of the tissue explantderived from a particular organism under conditions suitable to modulatethe expression of the CHRM3 gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of theCHRM3 gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a CHRM3 gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the CHRM3 gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the target RNA; and (b) introducing the siNAmolecule into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of theCHRM3 gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the CHRM3 gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one CHRM3 gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the CHRM3 genes; and (b) introducingthe siNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the CHRM3 genes in the tissue explant. In another embodiment, themethod further comprises introducing the tissue explant back into theorganism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the CHRM3 genes inthat organism.

In one embodiment, the invention features a method of modulating theexpression of a CHRM3 gene in an organism comprising: (a) synthesizingan siNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the CHRM3 gene; and (b) introducing the siNA molecule into theorganism under conditions suitable to modulate the expression of theCHRM3 gene in the organism. The level of CHRM3 protein or RNA can bedetermined as is known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one CHRM3 gene in an organism comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the CHRM3 genes; and (b) introducing the siNAmolecules into the organism under conditions suitable to modulate theexpression of the CHRM3 genes in the organism. The level of CHRM3protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a CHRM3 gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the CHRM3 gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the CHRM3 gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one CHRM3 gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the CHRM3 gene; and (b) contacting the cell invitro or in vivo with the siNA molecule under conditions suitable tomodulate the expression of the CHRM3 genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a CHRM3 gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the CHRM3 gene; and (b) contacting the cell ofthe tissue explant derived from a particular organism with the siNAmolecule under conditions suitable to modulate the expression of theCHRM3 gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the CHRM3 gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one CHRM3 gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the CHRM3 gene; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the CHRM3 genes in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of theCHRM3 genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a CHRM3 gene in an organism comprising: (a) synthesizingan siNA molecule of the invention, which can be chemically modified,wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the CHRM3 gene; and (b) introducing the siNAmolecule into the organism under conditions suitable to modulate theexpression of the CHRM3 gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one CHRM3 gene in an organism comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the CHRM3 gene; and (b) introducing the siNAmolecules into the organism under conditions suitable to modulate theexpression of the CHRM3 genes in the organism.

In one embodiment, the invention features a method of modulating theexpression of a CHRM3 gene in an organism comprising contacting theorganism with an siNA molecule of the invention under conditionssuitable to modulate the expression of the CHRM3 gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one CHRM3 gene in an organism comprisingcontacting the organism with one or more siNA molecules of the inventionunder conditions suitable to modulate the expression of the CHRM3 genesin the organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., CHRM3) gene expression through RNAi targeting of avariety of RNA molecules. In one embodiment, the siNA molecules of theinvention are used to target various RNAs corresponding to a targetgene. Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as CHRM3 family genes. As such, siNA molecules targetingmultiple CHRM3 targets can provide increased therapeutic effect. Inaddition, siNA can be used to characterize pathways of gene function ina variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for example,the progression and/or maintenance of cancer.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession Nos., for example CHRM3 genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(e.g., for an siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target CHRM3 RNA sequence. Inanother embodiment, the siNA molecules of (a) have strands of a fixedlength, for example about 23 nucleotides in length. In yet anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described in Example7 herein. In another embodiment, the assay can comprise a cell culturesystem in which target RNA is expressed. In another embodiment,fragments of CHRM3 RNA are analyzed for detectable levels of cleavage,for example by gel electrophoresis, Northern blot analysis, or RNAseprotection assays, to determine the most suitable target site(s) withinthe target CHRM3 RNA sequence. The target CHRM3 RNA sequence can beobtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 19 to about25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. Inone embodiment, the assay can comprise a reconstituted in vitro siNAassay as described herein. In another embodiment, the assay can comprisea cell culture system in which target RNA is expressed. Fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for reducing orpreventing tissue rejection in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thereduction or prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating aCHRM3 gene target, comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a CHRM3 target gene;(b) introducing the siNA molecule into a cell, tissue, or organism underconditions suitable for modulating expression of the CHRM3 target genein the cell, tissue, or organism; and (c) determining the function ofthe gene by assaying for any phenotypic change in the cell, tissue, ororganism.

In another embodiment, the invention features a method for validating aCHRM3 target comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a CHRM3 target gene;(b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the CHRM3 target genein the biological system; and (c) determining the function of the geneby assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, or organism, or extract thereof. The term biological system alsoincludes reconstituted RNAi systems that can be used in an in vitrosetting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically modified, that can beused to modulate the expression of a CHRM3 target gene in a biologicalsystem, including, for example, in a cell, tissue, or organism. Inanother embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically modified,that can be used to modulate the expression of more than one CHRM3target gene in a biological system, including, for example, in a cell,tissue, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for exampleunder hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications, for example, one or more chemical modificationshaving any of Formulae I-VII or any combination thereof that increasesthe nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siNA molecules having sequence homology to the chemicallymodified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically modified siNAmolecule.

In one embodiment, the invention features chemically modified siNAconstructs that mediate RNAi against CHRM3 in a cell, wherein thechemical modifications do not significantly effect the interaction ofsiNA with a target RNA molecule, DNA molecule and/or proteins or otherfactors that are essential for RNAi in a manner that would decrease theefficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against CHRM3 comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against CHRM3target RNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against CHRM3target DNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against CHRM3 with improved cellular uptake comprising(a) introducing nucleotides having any of Formula I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against CHRM3, wherein the siNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability, comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of an siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, an siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19” and “Stab 17/22”chemistries and variants thereof (see Table IV) wherein the 5′-end and3′-end of the sense strand of the siNA do not comprise a hydroxyl groupor phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of an siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19” and “Stab 17/22” chemistries andvariants thereof (see Table IV) wherein the 5′-end and 3′-end of thesense strand of the siNA do not comprise a hydroxyl group or phosphategroup.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex ordouble-stranded structure, for example wherein the double-strandedregion is about 19 base pairs); the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siNA is assembled froma single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single-stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single-stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide, chemicallymodified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), andothers. In addition, as used herein, the term RNAi is meant to beequivalent to other terms used to describe sequence specific RNAinterference, such as post transcriptional gene silencing, translationalinhibition, or epigenetics. For example, siNA molecules of the inventioncan be used to epigenetically silence genes at both thepost-transcriptional level and the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure to alter gene expression (see, for example, Verdelet al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science,303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218;and Hall et al., 2002, Science, 297, 2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003).

In one embodiment, an siNA molecule of the invention is amultifunctional siNA, (see for example FIGS. 16-22 and Jadhav et al.,U.S. Ser. No. 60/543,480, filed Feb. 10, 2004). The multifunctional siNAof the invention can comprise sequence targeting, for example, tworegions of CHRM3 RNA (see for example target sequences in Tables II andIII).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 19 to about 22 (e.g., about 19, 20, 21, or 22) nucleotides)and a loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 7,or 8) nucleotides, and a sense region having about 3 to about 18 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18)nucleotides that are complementary to the antisense region. Theasymmetric hairpin siNA molecule can also comprise a 5′-terminalphosphate group that can be chemically modified. The loop portion of theasymmetric hairpin siNA molecule can comprise nucleotides,non-nucleotides, linker molecules, or conjugate molecules as describedherein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 19 to about 22 (e.g. about 19, 20,21, or 22) nucleotides) and a sense region having about 3 to about 18(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18)nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (FRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Aberrant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of anorganism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts.

By “CHRM3” as used herein is meant, any cholinergic receptor muscarinic3 protein, peptide, or polypeptide, for example encoded by sequencesreferred to by accession numbers shown in Table I. The term “CHRM3” alsorefers to nucleic acid sequences encoding any cholinergic receptormuscarinic 3 protein, peptide, or polypeptide, such as CHRM3 RNA orCHRM3 DNA.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system or organism to anotherbiological system or organism. The polynucleotide can include bothcoding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence.

The siNA molecules of the invention represent a novel therapeuticapproach to treat a variety of disease and conditions such as chronicobstructive pulmonary disease or “COPD”, asthma, allergic rhinitis,sinusitis, pulmonary vasoconstriction, inflammation, allergies, impededrespiration, respiratory distress syndrome, cystic fibrosis, pulmonaryhypertension, pulmonary vasoconstriction, emphysema, irritable bowelsyndrome, Alzheimer's disease, urinary incontinence and any otherdiseases or conditions that are related to or will respond to the levelsof CHRM3 in a cell or tissue, alone or in combination with othertherapies. The reduction of CHRM3 expression and thus reduction in thelevel of the respective protein relieves, to some extent, the symptomsof the disease or condition.

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 18 to about 24nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,23, or 24 nucleotides in length. In another embodiment, the siNAduplexes of the invention independently comprise about 17 to about 23base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., 38, 39, 40,41, 42, 43 or 44) nucleotides in length and comprising about 16 to about22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNAmolecules of the invention are shown in Table II. Exemplary syntheticsiNA molecules of the invention are shown in Table III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through injection, infusion pump or stent, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in TablesII-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consistessentially of sequences defined in these tables and figures.Furthermore, the chemically modified constructs described in Table IVcan be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treatdiseases or conditions discussed herein (e.g., cancers and otherproliferative conditions). For example, to treat a particular disease orcondition, the siNA molecules can be administered to a subject or can beadministered to other appropriate cells evident to those skilled in theart, individually or in combination with one or more drugs underconditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to treat conditions or diseases discussedabove. For example, the described molecules could be used in combinationwith one or more known therapeutic agents to treat a disease orcondition. Non-limiting examples of other therapeutic agents that can bereadily combined with an siNA molecule of the invention are enzymaticnucleic acid molecules, allosteric nucleic acid molecules, antisense,decoy, or aptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules, and other organic and/or inorganiccompounds including metals, salts and ions.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. Theantisense strand of constructs A-F comprise sequence complementary toany target nucleic acid sequence of the invention. Furthermore, when aglyceryl moiety (L) is present at the 3′-end of the antisense strand forany construct shown in FIG. 4 A-F, the modified internucleotide linkageis optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a CHRM3 siNA sequence. Such chemicalmodifications can be applied to any CHRM3 sequence and/or CHRM3polymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined CHRM3 target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a CHRM3 targetsequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined CHRM3 target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palidrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double-stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palidrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double-strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH, HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes; nucleic acid molecules coupled with known smallmolecule modulators; or intermittent treatment with combinations ofmolecules, including different motifs and/or other chemical orbiological molecules). The treatment of subjects with siNA molecules canalso include combinations of different types of nucleic acid molecules,such as enzymatic nucleic acid molecules (ribozymes), allozymes,antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1 position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1 carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′—NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to treat, forexample, chronic obstructive pulmonary disease or “COPD”, asthma,allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation,allergies, impeded respiration, respiratory distress syndrome, cysticfibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema,irritable bowel syndrome, Alzheimer's disease, urinary incontinence andany other diseases or conditions that are related to or will respond tothe levels of CHRM3 in a cell or tissue, alone or in combination withother therapies. For example, an siNA molecule can comprise a deliveryvehicle, including liposomes, for administration to a subject, carriersand diluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations. Methods for the delivery of nucleic acidmolecules are described in Akhtar et al., 1992, Trends Cell Bio., 2,139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140;Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Leeet al., 2000, ACS Symp. Ser., 752, 184-192, all of which areincorporated herein by reference. Beigelman et al., U.S. Pat. No.6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. Alternatively, the nucleic acid/vehiclecombination is locally delivered by direct injection or by use of aninfusion pump.

In one embodiment, the nucleic acid molecules or the invention areadministered via pulmonary delivery, such as by inhalation of an aerosolor spray dried formulation administered by an inhalation device ornebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the nucleicacid compositions of the invention can optionally contain a dispersantwhich serves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which can be delivered by means of aninsufflator. In the insufflator, the powder, e.g., a metered dosethereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquefied propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example US PatentApplication No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;6,565,885.

In one embodiment, an siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes that lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes exposes the siNA molecules of theinvention to an accessible diseased tissue. The rate of entry of a druginto the circulation has been shown to be a function of molecular weightor size. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cells producing excess repeat expansion genes.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85);biodegradable polymers, such as poly(DL-lactide-coglycolide)microspheres for sustained release delivery (Emerich, D F et al, 1999,Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those madeof polybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly(ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 10/151,116, filed May 17, 2002. In one embodiment, nucleic acidmolecules of the invention are complexed with or covalently attached tonanoparticles, such as Hepatitis B virus S, M, or L envelope proteins(see for example Yamado et al., 2003, Nature Biotechnology, 21, 885). Inone embodiment, nucleic acid molecules of the invention are deliveredwith specificity for human tumor cells, specifically non-apoptotic humantumor cells including for example T-cells, hepatocytes, breast carcinomacells, ovarian carcinoma cells, melanoma cells, intestinal epithelialcells, prostate cells, testicular cells, non-small cell lung cancers,small cell lung cancers, etc.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi: 10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

CHRM3 Biology and Biochemistry

Cholinergic reflexes underlie respiratory constriction and mucosal glandsecretion and are mediated by muscarinic receptors. The muscariniccholinergic (acetylcholine) receptors belong to the larger family of Gprotein-coupled receptors. Muscarinic acetylcholine receptors in mammalsconsist of five subtypes (M1-M5) encoded by distinct genes. Thesereceptors are widely expressed throughout the body and play a variety ofroles in the peripheral and central nervous systems. The functionaldiversity of these receptors is defined by the ligand binding ofacetylcholine and includes cellular responses such as adenylate cyclaseinhibition, phosphoinositide degeneration, and modulation of potassiumchannels.

Muscarinic receptors influence many effects of acetylcholine in thecentral and peripheral nervous system. For example, the muscariniccholinergic receptor 3 controls smooth muscle contraction and itsstimulation results in secretion of glandular tissue in the respiratorytract. M3 receptor stimulation activates a pertussis toxin insensitiveG(q) protein that in turn activates phospholipase C (PLC) resulting inthe release of inositol trisphosphate (IP3) and diacylglycerol (DAG)(Spector Ed., 1999, Anticholinergic Agents in the Upper and LowerAirways, 1999, Volume 134, p 16-17). The release of IP3 mediates releaseof calcium whereas DAG activates protein kinase C, the combination ofwhich leads to smooth muscle contraction in airway, gastrointestinal,urinary bladder, vascular, and other tissues. M3 receptors aredistributed throughout the large and small airways and are highlylocalized to smooth muscle and submucosal gland cells. In theselocations, M3 receptor activity induces contraction in smooth musclecells and exocytosis in submucosal gland cells. Non-selective M3receptor antagonists such as atropine, ipratropium bromide and selectiveM3 receptor antagonists such as darifenacin and revatropate all inhibitcholinergially induced smooth muscle contraction and exocytosis fromsmooth muscle cells and submucosal gland cells respectively (SpectorEd., 1999, Anticholinergic Agents in the Upper and Lower Airways, 1999,Volume 134, p 16-17).

The use of small interfering nucleic acid molecules targeting CHRM3,therefore provides a class of novel therapeutic agents that can be usedin the treatment of diseases and conditions associated with airway M3receptor induced smooth muscle constriction and glandular secretion suchas chronic obstructive pulmonary disease or “COPD”, asthma, allergicrhinitis, sinusitis, irritable bowel syndrome, and any other diseases orconditions that are related to or will respond to the levels of CHRM3 ina cell or tissue (e.g. Alzheimer's disease, urinary incontinence andother conditions, see for example Choi et al., WO 01/029176), alone orin combination with other therapies.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H20 followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see for example Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a CHRM3target sequence is used to screen for target sites in cells expressingCHRM3 RNA, such as airway smooth muscle (ASM) or submucosal gland (e.g.,serous or mucus) cells. The general strategy used in this approach isshown in FIG. 9. A non-limiting example of such is a pool comprisingsequences having any of SEQ ID NOS 1-304. Cells expressing CHRM3 aretransfected with the pool of siNA constructs and cells that demonstratea phenotype associated with CHRM3 inhibition are sorted. The pool ofsiNA constructs can be expressed from transcription cassettes insertedinto appropriate vectors (see for example FIG. 7 and FIG. 8). The siNAfrom cells demonstrating a positive phenotypic change (e.g., decreasedproliferation, decreased CHRM3 mRNA levels or decreased CHRM3 proteinexpression), are sequenced to determine the most suitable target site(s)within the target CHRM3 RNA sequence.

Example 4 CHRM3 Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the CHRM3 RNAtarget and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Deprotection and purification of the siNA can beperformed as is generally described in Usman et al., U.S. Pat. No.5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellonet al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No.6,303,773, or Scaringe supra, incorporated by reference herein in theirentireties. Additionally, deprotection conditions can be modified toprovide the best possible yield and purity of siNA constructs. Forexample, applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting CHRM3 RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with CHRM3 target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriateCHRM3 expressing plasmid using T7 RNA polymerase or via chemicalsynthesis as described herein. Sense and antisense siNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug·mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²p] CTP, passed over aG 50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites theCHRM3 RNA target for siNA mediated RNAi cleavage, wherein a plurality ofsiNA constructs are screened for RNAi mediated cleavage of the CHRM3 RNAtarget, for example, by analyzing the assay reaction by electrophoresisof labeled target RNA, or by Northern blotting, as well as by othermethodology well known in the art.

Example 7 Nucleic Acid Inhibition of CHRM3 Target RNA In Vitro

siNA molecules targeted to the human CHRM3 RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within theCHRM3 RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting CHRM3.First, the reagents are tested in cell culture using, for example,airway smooth muscle (ASM) or submucosal gland (e.g., serous or mucus)cells, to determine the extent of RNA and protein inhibition. siNAreagents (e.g.; see Tables II and III) are selected against the CHRM3target as described herein. RNA inhibition is measured after delivery ofthese reagents by a suitable transfection agent to, for example, airwaysmooth muscle (ASM) or submucosal gland (e.g., serous or mucus) cells.Relative amounts of target RNA are measured versus actin using real-timePCR monitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparisonis made to a mixture of oligonucleotide sequences made to unrelatedtargets or to a randomized siNA control with the same overall length andchemistry, but randomly substituted at each position. Primary andsecondary lead reagents are chosen for the target and optimizationperformed. After an optimal transfection agent concentration is chosen,a RNA time-course of inhibition is performed with the lead siNAmolecule. In addition, a cell-plating format can be used to determineRNA inhibition.

Delivery of siNA to Cells

Cells (e.g., ASM or submucosal gland cells) are seeded, for example, at1×10⁵ cells per well of a six-well dish in EGM-2 (BioWhittaker) the daybefore transfection. siNA (final concentration, for example 20 nM) andcationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGMbasal media (Bio Whittaker) at 37° C. for 30 minutes in polystyrenetubes. Following vortexing, the complexed siNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TAQMAN® PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Animal Models Useful to Evaluate the Down-Regulation of CHRM3Gene Expression

Evaluating the efficacy of anti-CHRM3 agents in animal models is animportant prerequisite to human clinical trials. Dawkins et al., 2001,Thorax, 56, 972-977, provides a comprehensive review of animal models ofchronic obstructive pulmonary disease (COPD). Wright et al., 2002,Chest, 122, 301s-306s, describe an animal model of cigarette smokeinduced COPD. Zhang et al., 2004, J. Allergy Clin. Immunol, 113,320-326, describe an animal model of asthma that utilizes asthma likebiphasic airway responses in Brown Norway rats that are sensitized viadermal exposure to dry trimellitic anhydride powder. Wilder et al.,1999, Am. J. Respir. Cell Mol. Biol., 20, 1326-1334, describe murinemodels of allergic asthma induction. Grubb et al., 1999, Physiol. Rev.,79(1 Suppl), S193-214, provides a review of murine models of cysticfibrosis. Li et al., 2003, Reprod. Biol. Endocrinol., 1, 83, describes aferret model of cystic fibrosis and reviews other models of cysticfibrosis of various other animals including pig, sheep, and rabbits.Sugimoto et al., 2000, Immunopharmacology, 48, 1-7 and Shimizu et al.,2000, Am. J. Respir. Crit. Care Med., 161, 1648-54, describe rat modelsof allergic rhinitis. All of these models can be adapted for use forpre-clinical evaluation of the efficacy of nucleic acid compositions ofthe invention in modulating CHRM3 gene expression toward therapeuticuse.

Example 9 RNAi Mediated Inhibition of CHRM3 Expression in Cell Culture

Inhibition of CHRM3 RNA Expression Using siNA Targeting CHRM3 RNA

siNA constructs (Table III) are tested for efficacy in reducing CHRM3RNA expression in, for example, SH-SY5Y or HT29 cells. Cells are platedapproximately 24 hours before transfection in 96-well plates at5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells are 70-90% confluent. For transfection, annealedsiNAs are mixed with the transfection reagent (Lipofectamine 2000,Invitrogen) in a volume of 50 μl/well and incubated for 20 min. at roomtemperature. The siNA transfection mixtures are added to cells to give afinal siNA concentration of 25 nM in a volume of 150 μl. Each siNAtransfection mixture is added to 3 wells for triplicate siNA treatments.Cells are incubated at 37° for 24 h in the continued presence of thesiNA transfection mixture. At 24 h, RNA is prepared from each well oftreated cells. The supernatants with the transfection mixtures are firstremoved and discarded, then the cells are lysed and RNA prepared fromeach well. Target gene expression following treatment is evaluated byRT-PCR for the target gene and for a control gene (36B4, an RNApolymerase subunit) for normalization. The triplicate data is averagedand the standard deviations determined for each treatment. Normalizeddata are graphed and the percent reduction of target mRNA by activesiNAs in comparison to their respective inverted control siNAs isdetermined.

Example 10 Indications

The present body of knowledge in CHRM3 research indicates the need formethods to assay CHRM3 activity and for compounds that can regulateCHRM3 expression for research, diagnostic, and therapeutic use. Asdescribed herein, the nucleic acid molecules of the present inventioncan be used in assays to diagnose disease state related of CHRM3 levels.In addition, the nucleic acid molecules can be used to treat diseasestate related to CHRM3 levels.

Particular conditions and disease states that can be associated withCHRM3 expression modulation include, but are not limited to chronicobstructive pulmonary disease or “COPD”, asthma, allergic rhinitis,sinusitis, pulmonary vasoconstriction, inflammation, allergies, impededrespiration, respiratory distress syndrome, cystic fibrosis, pulmonaryhypertension, pulmonary vasoconstriction, emphysema, irritable bowelsyndrome, Alzheimer's disease, urinary incontinence and any otherdiseases or conditions that are related to or will respond to the levelsof CHRM3 in a cell or tissue, alone or in combination with othertherapies.

The use of anticholinergic agents, anti-inflammatories, bronchodilators,adenosine inhibitors, adenosine A1 receptor inhibitors, non-selective M3receptor antagonists such as atropine, ipratropium bromide and selectiveM3 receptor antagonists such as darifenacin and revatropate are allnon-limiting examples of agents that can be combined with or used inconjunction with the nucleic acid molecules (e.g. siNA molecules) of theinstant invention. Those skilled in the art will recognize that othercompounds and therapies used to treat the diseases and conditionsdescribed herein can similarly be combined with the nucleic acidmolecules of the instant invention (e.g. siNA molecules) and are hencewithin the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I CHRM3 Accession Numbers NM_000740 Homo sapiens cholinergicreceptor, muscarinic 3 (CHRM3), mRNAgi|4502818|ref|NM_000740.1|[4502818] U29589 Human m3 muscarinicacetylcholine receptor (CHRM3) gene, complete cdsgi|903978|gb|U29589.1|HSU29589[903978] X15266 Homo sapiens gene formuscarinic acetylcholine receptor HM3gi|32323|emb|X15266.1|HSACM3[32323] AB041395 Homo sapiens CHRM3 gene formuscarinic acetylcholine receptor m3, complete cdsgi|7592980|dbj|AB041395.1|[7592980] AF498917 Homo sapiens muscarinicacetylcholine receptor M3 (CHRM3) mRNA, complete cdsgi|20302972|gb|AF498917.1|[20302972] AK074575 Homo sapiens cDNA FLJ90094fis, clone HEMBA1005545, highly similar to MUSCARINIC ACETYLCHOLINERECEPTOR M3 gi|22760104|dbj|AK074575.1|[22760104] AF385589 Homo sapiensm3 muscarinic cholinergic receptor (CHRM3) mRNA, partial cdsgi|14573540|gb|AF385589.1|AF385589[14573540] AF279779 Homo sapiens cloneN10 NTera2D1 teratocarcinoma, m3 muscarinic acetylcholine receptor mRNA,partial cds gi|11095820|gb|AF279779.1|AF279779[11095820]

TABLE II CHRM3 siNA and Target Sequences CHRM3 NM_000740.1 Seq Seq SeqPos Seq ID UPos Upper seq ID LPos Lower seq ID 3 GACCUUGCACAAUAACAGU 1 3GACCUUGCACAAUAACAGU 1 21 ACUGUUAUUGUGCAAGGUC 100 21 UACAACCUCGCCUUUGUUU2 21 UACAACCUCGCCUUUGUUU 2 39 AAACAAAGGCGAGGUUGUA 101 39UCCAAACAUCAGCUCCUCC 3 39 UCCAAACAUCAGCUCCUCC 3 57 GGAGGAGCUGAUGUUUGGA102 57 CUGGAUACACAGCCCCUCC 4 57 CUGGAUACACAGCCCCUCC 4 75GGAGGGGCUGUGUAUCCAG 103 75 CGAUGCAGGGCUGCCCCCG 5 75 CGAUGCAGGGCUGCCCCCG5 93 CGGGGGCAGCCCUGCAUCG 104 93 GGGAACCGUCACUCAUUUC 6 93GGGAACCGUCACUCAUUUC 6 111 GAAAUGAGUGACGGUUCCC 105 111CGGCAGCUACAAUGUUUCU 7 111 CGGCAGCUACAAUGUUUCU 7 129 AGAAACAUUGUAGCUGCCG106 129 UCGAGCAGCUGGCAAUUUC 8 129 UCGAGCAGCUGGCAAUUUC 8 147GAAAUUGCCAGCUGCUCGA 107 147 CUCCUCUCCAGACGGUACC 9 147CUCCUCUCCAGACGGUACC 9 165 GGUACCGUCUGGAGAGGAG 108 165CACCGAUGACCCUCUGGGA 10 165 CACCGAUGACCCUCUGGGA 10 183UCCCAGAGGGUCAUCGGUG 109 183 AGGUCAUACCGUCUGGCAA 11 183AGGUCAUACCGUCUGGCAA 11 201 UUGCCAGACGGUAUGACCU 110 201AGUGGUCUUCAUCGCUUUC 12 201 AGUGGUCUUCAUCGCUUUC 12 219GAAAGCGAUGAAGACCACU 111 219 CUUAACGGGCAUCCUGGCC 13 219CUUAACGGGCAUCCUGGCC 13 237 GGCCAGGAUGCCCGUUAAG 112 237CUUGGUGACCAUCAUCGGC 14 237 CUUGGUGACCAUCAUCGGC 14 255GCCGAUGAUGGUCACCAAG 113 255 CAACAUCCUGGUAAUUGUG 15 255CAACAUCCUGGUAAUUGUG 15 273 CACAAUUACCAGGAUGUUG 114 273GUCAUUUAAGGUCAACAAG 16 273 GUCAUUUAAGGUCAACAAG 16 291CUUGUUGACCUUAAAUGAC 115 291 GCAGCUGAAGACGGUCAAC 17 291GCAGCUGAAGACGGUCAAC 17 309 GUUGACCGUCUUCAGCUGC 116 309CAACUACUUCCUCUUAAGC 18 309 CAACUACUUCCUCUUAAGC 18 327GCUUAAGAGGAAGUAGUUG 117 327 COUGGOCUGUGOOGAUCUG 19 327CCUGGCCUGUGCCGAUCUG 19 345 CAGAUCGGCACAGGCCAGG 118 345GAUUAUCGGGGUCAUUUCA 20 345 GAUUAUCGGGGUCAUUUCA 20 363UGAAAUGACCCCGAUAAUC 119 363 AAUGAAUCUGUUUACGACC 21 363AAUGAAUCUGUUUACGACC 21 381 GGUCGUAAACAGAUUCAUU 120 381CUACAUCAUCAUGAAUCGA 22 381 CUACAUCAUCAUGAAUCGA 22 399UCGAUUCAUGAUGAUGUAG 121 399 AUGGGCCUUAGGGAACUUG 23 399AUGGGCCUUAGGGAACUUG 23 417 CAAGUUCCCUAAGGCCCAU 122 417GGCCUGUGACCUCUGGCUU 24 417 GGCCUGUGACCUCUGGCUU 24 435AAGCCAGAGGUCACAGGCC 123 435 UGCCAUUGACUACGUAGCC 25 435UGCCAUUGACUACGUAGCC 25 453 GGCUACGUAGUCAAUGGCA 124 453CAGCAAUGCCUCUGUUAUG 26 453 CAGCAAUGCCUCUGUUAUG 26 471CAUAACAGAGGCAUUGCUG 125 471 GAAUCUUCUGGUCAUCAGC 27 471GAAUCUUCUGGUCAUCAGC 27 489 GCUGAUGACCAGAAGAUUC 126 489CUUUGACAGAUACUUUUCC 28 489 CUUUGACAGAUACUUUUCC 28 507GGAAAAGUAUCUGUCAAAG 127 507 CAUCACGAGGCCGCUCACG 29 507CAUCACGAGGCCGCUCACG 29 525 CGUGAGCGGCCUCGUGAUG 128 525GUACCGAGCCAAACGAACA 30 525 GUACCGAGCCAAACGAACA 30 543UGUUCGUUUGGCUCGGUAC 129 543 AACAAAGAGAGCCGGUGUG 31 543AACAAAGAGAGCCGGUGUG 31 561 CACACCGGCUCUCUUUGUU 130 561GAUGAUCGGUCUGGCUUGG 32 561 GAUGAUCGGUCUGGCUUGG 32 579CCAAGCCAGACCGAUCAUC 131 579 GGUCAUCUCCUUUGUCCUU 33 579GGUCAUCUCCUUUGUCCUU 33 597 AAGGACAAAGGAGAUGACC 132 597UUGGGCUCCUGCCAUCUUG 34 597 UUGGGCUCCUGCCAUCUUG 34 615CAAGAUGGCAGGAGCCCAA 133 615 GUUCUGGCAAUACUUUGUU 35 615GUUCUGGCAAUACUUUGUU 35 633 AACAAAGUAUUGCCAGAAC 134 633UGGAAAGAGAACUGUGCCU 36 633 UGGAAAGAGAACUGUGCCU 36 651AGGCACAGUUCUCUUUCCA 135 651 UCCGGGAGAGUGCUUCAUU 37 651UCCGGGAGAGUGCUUCAUU 37 669 AAUGAAGCACUCUCCCGGA 136 669UCAGUUCCUCAGUGAGCCC 38 669 UCAGUUCCUCAGUGAGCCC 38 687GGGCUCACUGAGGAACUGA 137 687 CACCAUUACUUUUGGCACA 39 687CACCAUUACUUUUGGCACA 39 705 UGUGCCAAAAGUAAUGGUG 138 705AGCCAUCGCUGCUUUUUAU 40 705 AGCCAUCGCUGCUUUUUAU 40 723AUAAAAAGCAGCGAUGGCU 139 723 UAUGCCUGUCACCAUUAUG 41 723UAUGCCUGUCACCAUUAUG 41 741 CAUAAUGGUGACAGGCAUA 140 741GACUAUUUUAUACUGGAGG 42 741 GACUAUUUUAUACUGGAGG 42 759CCUCCAGUAUAAAAUAGUC 141 759 GAUCUAUAAGGAAACUGAA 43 759GAUCUAUAAGGAAACUGAA 43 777 UUCAGUUUCCUUAUAGAUC 142 777AAAGCGUACCAAAGAGCUU 44 777 AAAGCGUACCAAAGAGCUU 44 795AAGCUCUUUGGUACGCUUU 143 795 UGCUGGCCUGCAAGCCUCU 45 795UGCUGGCCUGCAAGCCUCU 45 813 AGAGGCUUGCAGGCCAGCA 144 813UGGGACAGAGGCAGAGACA 46 813 UGGGACAGAGGCAGAGACA 46 831UGUCUCUGOCUCUGUCOCA 145 831 AGAAAACUUUGUCCACCCC 47 831AGAAAACUUUGUCCACCCC 47 849 GGGGUGGACAAAGUUUUCU 146 849CACGGGCAGUUCUCGAAGC 48 849 CACGGGCAGUUCUCGAAGC 48 867GCUUCGAGAACUGCCCGUG 147 867 CUGCAGCAGUUACGAACUU 49 867CUGCAGCAGUUACGAACUU 49 885 AAGUUCGUAACUGCUGCAG 148 885UCAACAGCAAAGCAUGAAA 50 885 UCAACAGCAAAGCAUGAAA 50 903UUUCAUGCUUUGCUGUUGA 149 903 ACGCUCCAACAGGAGGAAG 51 903ACGCUCCAACAGGAGGAAG 51 921 CUUCCUCCUGUUGGAGCGU 150 921GUAUGGCCGCUGCCACUUC 52 921 GUAUGGCCGCUGCCACUUC 52 939GAAGUGGCAGCGGCCAUAC 151 939 CUGGUUCACAACCAAGAGC 53 939CUGGUUCACAACCAAGAGC 53 957 GCUCUUGGUUGUGAACCAG 152 957CUGGAAACCCAGCUCCGAG 54 957 CUGGAAACCCAGCUCCGAG 54 975CUCGGAGCUGGGUUUCCAG 153 975 GCAGAUGGACCAAGACCAC 55 975GCAGAUGGACCAAGACCAC 55 993 GUGGUCUUGGUCCAUCUGC 154 993CAGCAGCAGUGACAGUUGG 56 993 CAGCAGCAGUGACAGUUGG 56 1011CCAACUGUCACUGCUGCUG 155 1011 GAACAACAAUGAUGCUGCU 57 1011GAACAACAAUGAUGCUGCU 57 1029 AGCAGCAUCAUUGUUGUUC 156 1029UGCCUCCCUGGAGAACUCC 58 1029 UGCCUCCCUGGAGAACUCC 58 1047GGAGUUCUCCAGGGAGGCA 157 1047 CGCCUCCUCCGACGAGGAG 59 1047CGCCUCCUCCGACGAGGAG 59 1065 CUCCUCGUCGGAGGAGGCG 158 1065GGACAUUGGCUCCGAGACG 60 1065 GGACAUUGGCUCCGAGACG 60 1083CGUCUCGGAGCCAAUGUCC 159 1083 GAGAGCCAUCUACUCCAUC 61 1083GAGAGCCAUCUACUCCAUC 61 1101 GAUGGAGUAGAUGGCUCUC 160 1101CGUGCUCAAGCUUCCGGGU 62 1101 CGUGCUCAAGCUUCCGGGU 62 1119ACCCGGAAGCUUGAGCACG 161 1119 UCACAGCACCAUCCUCAAC 63 1119UCACAGCACCAUCCUCAAC 63 1137 GUUGAGGAUGGUGCUGUGA 162 1137CUCCACCAAGUUACCCUCA 64 1137 CUCCACCAAGUUACCCUCA 64 1155UGAGGGUAACUUGGUGGAG 163 1155 AUCGGACAACCUGCAGGUG 65 1155AUCGGACAACCUGCAGGUG 65 1173 CACCUGCAGGUUGUCCGAU 164 1173GCCUGAGGAGGAGCUGGGG 66 1173 GCCUGAGGAGGAGCUGGGG 66 1191CCCCAGCUCCUCCUCAGGC 165 1191 GAUGGUGGACUUGGAGAGG 67 1191GAUGGUGGACUUGGAGAGG 67 1209 CCUCUCCAAGUCCACCAUC 166 1209GAAAGCCGACAAGCUGCAG 68 1209 GAAAGCCGACAAGCUGCAG 68 1227CUGCAGCUUGUCGGCUUUC 167 1227 GGCCCAGAAGAGCGUGGAC 69 1227GGCCCAGAAGAGCGUGGAC 69 1245 GUCCACGCUCUUCUGGGCC 168 1245CGAUGGAGGCAGUUUUCCA 70 1245 CGAUGGAGGCAGUUUUCCA 70 1263UGGAAAACUGCCUCCAUCG 169 1263 AAAAAGCUUCUCCAAGCUU 71 1263AAAAAGCUUCUCCAAGCUU 71 1281 AAGCUUGGAGAAGCUUUUU 170 1281UCCCAUCCAGCUAGAGUCA 72 1281 UCCCAUCCAGCUAGAGUCA 72 1299UGACUCUAGCUGGAUGGGA 171 1299 AGCCGUGGACACAGCUAAG 73 1299AGCCGUGGACACAGCUAAG 73 1317 CUUAGCUGUGUCCACGGCU 172 1317GACUUCUGACGUCAACUCC 74 1317 GACUUCUGACGUCAACUCC 74 1335GGAGUUGACGUCAGAAGUC 173 1335 CUCAGUGGGUAAGAGCACG 75 1335CUCAGUGGGUAAGAGCACG 75 1353 CGUGCUCUUACCCACUGAG 174 1353GGCCACUCUACCUCUGUCC 76 1353 GGCCACUCUACCUCUGUCC 76 1371GGACAGAGGUAGAGUGGCC 175 1371 CUUCAAGGAAGCCACUCUG 77 1371CUUCAAGGAAGCCACUCUG 77 1389 CAGAGUGGCUUCCUUGAAG 176 1389GGCCAAGAGGUUUGCUCUG 78 1389 GGCCAAGAGGUUUGCUCUG 78 1407CAGAGCAAACCUCUUGGCC 177 1407 GAAGACCAGAAGUCAGAUC 79 1407GAAGACCAGAAGUCAGAUC 79 1425 GAUCUGACUUCUGGUCUUC 178 1425CACUAAGCGGAAAAGGAUG 80 1425 CACUAAGCGGAAAAGGAUG 80 1443CAUCCUUUUCCGCUUAGUG 179 1443 GUCCCUGGUCAAGGAGAAG 81 1443GUCCCUGGUCAAGGAGAAG 81 1461 CUUCUCCUUGACCAGGGAC 180 1461GAAAGCGGCCCAGACCCUC 82 1461 GAAAGCGGCCCAGACCCUC 82 1479GAGGGUCUGGGCCGCUUUC 181 1479 CAGUGCGAUCUUGCUUGCC 83 1479CAGUGCGAUCUUGCUUGCC 83 1497 GGCAAGCAAGAUCGCACUG 182 1497CUUCAUCAUCACUUGGACC 84 1497 CUUCAUCAUCACUUGGACC 84 1515GGUCCAAGUGAUGAUGAAG 183 1515 CCCAUACAACAUCAUGGUU 85 1515CCCAUACAACAUCAUGGUU 85 1533 AACCAUGAUGUUGUAUGGG 184 1533UCUGGUGAACACCUUUUGU 86 1533 UCUGGUGAACACCUUUUGU 86 1551ACAAAAGGUGUUCACCAGA 185 1551 UGACAGCUGCAUACCCAAA 87 1551UGACAGCUGCAUACCCAAA 87 1569 UUUGGGUAUGCAGCUGUCA 186 1569AACCUUUUGGAAUCUGGGC 88 1569 AACCUUUUGGAAUCUGGGC 88 1587GCCCAGAUUCCAAAAGGUU 187 1587 CUACUGGCUGUGCUACAUC 89 1587CUACUGGCUGUGCUACAUC 89 1605 GAUGUAGOACAGOCAGUAG 188 1605CAACAGCACCGUGAACCCC 90 1605 CAACAGCACCGUGAACCCC 90 1623GGGGUUCACGGUGCUGUUG 189 1623 CGUGUGCUAUGCUCUGUGC 91 1623CGUGUGCUAUGCUCUGUGC 91 1641 GCACAGAGCAUAGCACACG 190 1641CAACAAAACAUUCAGAACC 92 1641 CAACAAAACAUUCAGAACC 92 1659GGUUCUGAAUGUUUUGUUG 191 1659 CACUUUCAAGAUGCUGCUG 93 1659CACUUUCAAGAUGCUGCUG 93 1677 CAGCAGCAUCUUGAAAGUG 192 1677GCUGUGCCAGUGUGACAAA 94 1677 GCUGUGCCAGUGUGACAAA 94 1695UUUGUCACACUGGCACAGC 193 1695 AAAAAAGAGGCGCAAGCAG 95 1695AAAAAAGAGGCGCAAGCAG 95 1713 CUGCUUGCGCCUCUUUUUU 194 1713GCAGUACCAGCAGAGACAG 96 1713 GCAGUACCAGCAGAGACAG 96 1731CUGUCUCUGCUGGUACUGC 195 1731 GUCGGUCAUUUUUCACAAG 97 1731GUCGGUCAUUUUUCACAAG 97 1749 CUUGUGAAAAAUGACCGAC 196 1749GCGCGCACCCGAGCAGGCC 98 1749 GCGCGCACCCGAGCAGGCC 98 1767GGCCUGCUCGGGUGCGCGC 197 1753 GCACCCGAGCAGGCCUUGU 99 1753GCACCCGAGCAGGCCUUGU 99 1771 ACAAGGCCUGCUCGGGUGC 198 The 3′-ends of theUpper sequence and the Lower sequence of the siNA construct can includean overhang sequence, for example about 1, 2, 3, or 4 nucleotides inlength, preferably 2 nucleotides in length, wherein the overhangingsequence of the lower sequence is optionally complementary to a portionof the target sequence. The overhang can comprise the general structureB, BNN, NN, BNsN, or NsN, where B stands for any terminal cap moiety, Nstands for any nucleotide (e.g., thymidine) and s stands forphosphorothioate or other internucleotide linkage as described herein(e.g. internucleotide linkage having Formula I). The upper sequence isalso referred to as the sense strand, whereas the lower sequence is alsoreferred to as the antisense strand. The upper and lower sequences inthe Table can further comprise a chemical modification having FormulaeI-VII or any combination thereof (see for example chemical modificationsas shown in Table V herein).

TABLE III CHRM3 synthetic siNA and Target Sequences Target Seq Seq PosTarget Sequence ID SIRNA# Aliases Sequence ID 15 UAACAGUACAACCUCGCCUUUGU199 CHRM3:17U21 siNA sense ACAGUACAACCUCGCCUUUTT 207 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:21U21 siNA sense UACAACCUCGCCUUUGUUUTT208 313 UACUUCCUCUUAAGCCUGGCCUG 201 CHRM3:315U21 siNA senseCUUCCUCUUAAGCCUGGCCTT 209 315 CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:317U21siNA sense UCCUCUUAAGCCUGGCCUGTT 210 973 GAGCAGAUGGACCAAGACCACAG 203CHRM3:975U21 siNA sense GCAGAUGGACCAAGACCACTT 211 1710GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:1712U21 siNA senseAGCAGUACCAGCAGAGACATT 212 1712 AGCAGUACCAGCAGAGACAGUCG 205 CHRM3:1714U21siNA sense CAGUACCAGCAGAGACAGUTT 213 1713 GCAGUACCAGCAGAGACAGUCGG 206CHRM3:1715U21 siNA sense AGUACCAGCAGAGACAGUCTT 214 15UAACAGUACAACCUCGCCUUUGU 199 CHRM3:35L21 siNA (17C) AAAGGCGAGGUUGUACUGUTT215 antisense 19 AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:39L21 siNA (21C)AAACAAAGGCGAGGUUGUATT 216 antisense 313 UACUUCCUCUUAAGCCUGGCCUG 201CHRM3:333L21 siNA (315C) GGCCAGGCUUAAGAGGAAGTT 217 antisense 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:335L21 siNA (317C)CAGGCCAGGCUUAAGAGGATT 218 antisense 973 GAGCAGAUGGACCAAGACCACAG 203CHRM3:993L21 siNA (975C) GUGGUCUUGGUCCAUCUGCTT 219 antisense 1710GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:1730L21 siNA (1712C)UGUCUCUGCUGGUACUGCUTT 220 antisense 1712 AGCAGUACCAGCAGAGACAGUCG 205CHRM3:1732L21 siNA (1714C) ACUGUCUCUGCUGGUACUGTT 221 antisense 1713GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1733L21 siNA (1715C)GACUGUCUCUGCUGGUACUTT 222 antisense 15 UAACAGUACAACCUCGCCUUUGU 199CHRM3:17U21 siNA stab04 sense B AcAGuAcAAccucGccuuuTT B 223 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:21U21 siNA stab04 sense BuAcAAccucGccuuuGuuuTT B 224 313 UACUUCCUCUUAAGCCUGGCCUG 201 CHRM3:315U21siNA stab04 sense B cuuccucuuAAGccuGGccTT B 225 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:317U21 siNA stab04 sense BuccucuuAAGccuGGccuGTT B 226 973 GAGCAGAUGGACCAAGACCACAG 203 CHRM3:975U21siNA stab04 sense B GcAGAuGGAccAAGAccAcTT B 227 1710GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:1712U21 siNA stab04 sense BAGcAGuAccAGcAGAGAcATT B 228 1712 AGCAGUACCAGCAGAGACAGUCG 205CHRM3:1714U21 siNA stab04 sense B cAGuAccAGcAGAGAcAGuTT B 229 1713GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1715U21 siNA stab04 sense BAGuAccAGcAGAGAcAGucTT B 230 15 UAACAGUACAACCUCGCCUUUGU 199 CHRM3:35L21siNA (17C) stab05 AAAGGcGAGGuuGuAcuGuTsT 231 antisense 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:39L21 siNA (21C) stab05AAAcAAAGGcGAGGuuGuATsT 232 antisense 313 UACUUCCUCUUAAGCCUGGCCUG 201CHRM3:333L21 siNA (315C) stab05 GGccAGGcuuAAGAGGAAGTsT 233 antisense 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:335L21 siNA (317C) stab05cAGGccAGGcuuAAGAGGATsT 234 antisense 973 GAGCAGAUGGACCAAGACCACAG 203CHRM3:993L21 siNA (975C) stab05 GuGGucuuGGuccAucuGcTsT 235 antisense1710 GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:173CL21 siNA (1712C)uGucucuGcuGGuAcuGcuTsT 236 stab05 antisense 1712 AGCAGUACCAGCAGAGACAGUCG205 CHRM3:1732L21 siNA (1714C) AcuGucucuGcuGGuAcuGTsT 237 stab05antisense 1713 GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1733L21 siNA (1715C)GAcuGucucuGcuGGuAcuTsT 238 stab05 antisense 15 UAACAGUACAACCUCGCCUUUGU199 CHRM3:17U21 siNA stab07 sense B AcAGuAcAAccucGccuuuTT B 239 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:21U21 siNA stab07 sense BuAcAAccucGccuuuGuuuTT B 240 313 UACUUCCUCUUAAGCCUGGCCUG 201 CHRM3:315U21siNA stab07 sense B cuuccucuuAAGccuGGccTT B 241 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:317U21 siNA stab07 sense BuccucuuAAGccuGGccuGTT B 242 973 GAGCAGAUGGACCAAGACCACAG 203 CHRM3:975U21siNA stab07 sense B GcAGAuGGAccAAGAccAcTT B 243 1710GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:1712U21 siNA stab07 sense BAGcAGuAccAGcAGAGAcATT B 244 1712 AGCAGUACCAGCAGAGACAGUCG 205CHRM3:1714U21 siNA stab07 sense B cAGuAccAGcAGAGAcAGuTT B 245 1713GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1715U21 siNA stab07 sense BAGuAccAGcAGAGAcAGucTT B 246 15 UAACAGUACAACCUCGCCUUUGU 199 CHRM3:35L21siNA (17C) stab11 AAAGGcGAGGuuGuAcuGuTsT 247 antisense 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:39L21 siNA (21C) stab11AAAcAAAGGcGAGGuuGuATsT 248 antisense 313 UACUUCCUCUUAAGCCUGGCCUG 201CHRM3:333L21 siNA (315C) stab11 GGccAGGcuuAAGAGGAAGTsT 249 antisense 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:335L21 siNA (317C) stab11cAGGccAGGcuuAAGAGGATsT 250 antisense 973 GAGCAGAUGGACCAAGACCACAG 203CHRM3:993L21 siNA (975C) stab11 GuGGucuuGGuccAucuGcTsT 251 antisense1710 GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:173CL21 siNA (1712C)uGucucuGcuGGuAcuGcuTsT 252 stab11 antisense 1712 AGCAGUACCAGCAGAGACAGUCG205 CHRM3:1732L21 siNA (1714C) AcuGucucuGcuGGuAcuGTsT 253 stab11antisense 1713 GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1733L21 siNA (1715C)GAcuGucucuGcuGGuAcuTsT 254 stab11 antisense 15 UAACAGUACAACCUCGCCUUUGU199 CHRM3:17U21 siNA stab18 sense B AcAGuAcAAccucGccuuuTT B 255 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:21U21 siNA stab18 sense BuAcAAccucGccuuuGuuuTT B 256 313 UACUUCCUCUUAAGCCUGGCCUG 201 CHRM3:315U21siNA stab18 sense B cuuccucuuAAGccuGGccTT B 257 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:317U21 siNA stab18 sense BuccucuuAAGccuGGccuGTT B 258 973 GAGCAGAUGGACCAAGACCACAG 203 CHRM3:975U21siNA stab18 sense B GcAGAuGGAccAAGAccAcTT B 259 1710GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:1712U21 siNA stab18 sense BAGcAGuAccAGcAGAGAcATT B 260 1712 AGCAGUACCAGCAGAGACAGUCG 205CHRM3:1714U21 siNA stab18 sense B cAGuAccAGcAGAGAcAGuTT B 261 1713GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1715U21 siNA stab18 sense BAGuAccAGcAGAGAcAGucTT B 262 15 UAACAGUACAACCUCGCCUUUGU 199 CHRM3:35L21siNA (17C) stab08 AAAGGcGAGGuuGuAcuGuTsT 263 antisense 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:39L21 siNA (21C) stab08AAAcAAAGGcGAGGuuGuATsT 264 antisense 313 UACUUCCUCUUAAGCCUGGCCUG 201CHRM3:333L21 siNA (315C) stab08 GGccAGGcuuAAGAGGAAGTsT 265 antisense 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:335L21 siNA (317C) stab08cAGGccAGGcuuAAGAGGATsT 266 antisense 973 GAGCAGAUGGACCAAGACCACAG 203CHRM3:993L21 siNA (975C) stab08 GuGGucuuGGuccAucuGcTsT 267 antisense1710 GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:173CL21 siNA (1712C)uGucucuGcuGGuAcuGcuTsT 268 stab08 antisense 1712 AGCAGUACCAGCAGAGACAGUCG205 CHRM3:1732L21 siNA (1714C) AcuGucucuGcuGGuAcuGTsT 269 stab08antisense 1713 GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1733L21 siNA (1715C)GAcuGucucuGcuGGuAcuTsT 270 stab08 antisense 15 UAACAGUACAACCUCGCCUUUGU199 CHRM3:17U21 siNA stab09 sense B ACAGUACAACCUCGCCUUUTT B 271 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:21 U21 siNA stab09 sense BUACAACCUCGCCUUUGUUUTT B 272 313 UACUUCCUCUUAAGCCUGGCCUG 201 CHRM3:315U21siNA stab09 sense B CUUCCUCUUAAGCCUGGCCTT B 273 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:317U21 siNA stab09 sense BUCCUCUUAAGCCUGGCCUGTT B 274 973 GAGCAGAUGGACCAAGACCACAG 203 CHRM3:975U21siNA stab09 sense B GCAGAUGGACCAAGACCACTT B 275 1710GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:1712U21 siNA stab09 sense BAGCAGUACCAGCAGAGACATT B 276 1712 AGCAGUACCAGCAGAGACAGUCG 205CHRM3:1714U21 siNA stab09 sense B CAGUACCAGCAGAGACAGUTT B 277 1713GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1715U21 siNA stab09 sense BAGUACCAGCAGAGACAGUCTT B 278 15 UAACAGUACAACCUCGCCUUUGU 199 CHRM3:35L21siNA (17C) stab10 AAAGGCGAGGUUGUACUGUTsT 279 antisense 19AGUACAACCUCGCCUUUGUUUCC 200 CHRM3:39L21 siNA (21C) stab10AAACAAAGGCGAGGUUGUATsT 280 antisense 313 UACUUCCUCUUAAGCCUGGCCUG 201CHRM3:333L21 siNA (315C) stab10 GGCCAGGCUUAAGAGGAAGTsT 281 antisense 315CUUCCUCUUAAGCCUGGCCUGUG 202 CHRM3:335L21 siNA (317C) stab10CAGGCCAGGCUUAAGAGGATsT 282 antisense 973 GAGCAGAUGGACCAAGACCACAG 203CHRM3:993L21 siNA (975C) stab10 GUGGUCUUGGUCCAUCUGCTsT 283 antisense1710 GCAGCAGUACCAGCAGAGACAGU 204 CHRM3:173CL21 siNA (1712C)UGUCUCUGCUGGUACUGCUTsT 284 stab10 antisense 1712 AGCAGUACCAGCAGAGACAGUCG205 CHRM3:1732L21 siNA (1714C) ACUGUCUCUGCUGGUACUGTsT 285 stab10antisense 1713 GCAGUACCAGCAGAGACAGUCGG 206 CHRM3:1733L21 siNA (1715C)GACUGUCUCUGCUGGUACUTsT 286 stab10 antisense

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at S/AS 3′-ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually AS linkages“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and — Usually S 3′-ends “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and — Usually S 3′-ends“Stab 7” 2′-fluoro 2′-deoxy 5′ and — Usually S 3′-ends “Stab 8”2′-fluoro 2′-O- — 1 at 3′-end Usually AS Methyl “Stab 9” Ribo Ribo 5′and — Usually S 3′-ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS“Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12”2′-fluoro LNA 5′ and Usually S 3′-ends “Stab 13” 2′-fluoro LNA 1 at3′-end Usually AS “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end“Stab 16 Ribo 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 17”2′-O-Methyl 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 18” 2′-fluoro2′-O- 5′ and 1 at 3′-end Usually S Methyl 3′-ends “Stab 19” 2′-fluoro2′-O- 3′-end Usually AS Methyl “Stab 20” 2′-fluoro 2′-deoxy 3′-endUsually AS “Stab 21” 2′-fluoro Ribo 3′-end Usually AS “Stab 22” RiboRibo 3′-end — Usually AS CAP = any terminal cap, see for example FIG.10. All Stab 1-22 chemistries can comprise 3′-terminal thymidine (TT)residues All Stab 1-22 chemistries typically comprise about 21nucleotides, but can vary as described herein. S = sense strand AS =antisense strand

TABLE V Wait Time* Wait Time* Wait Time* Reagent Equivalents Amount DNA2′-O-methyl RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: DNA/2′- Amount: DNA/2′-O- Wait Time* Wait Time*Wait Time* Reagent O-methyl/Ribo methyl/Ribo DNA 2′-O-methyl RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified short interfering nucleic acid molecule (siNA),wherein: (a) the siNA molecule comprises a sense strand and a separateantisense strand, each strand having one or more pyrimidine nucleotidesand one or more purine nucleotides; (b) each strand is independently 18to 27 nucleotides in length, and together comprise a duplex havingbetween 18 and 23 base pairs; (c) the antisense strand is complementaryto a human cholinergic receptor muscarinic 3 (CHRM3) RNA sequencecomprising SEQ ID NO: 305; (d) each pyrimidine nucleotide of the sensestrand has a 2′-fluoro modification, each purine nucleotide of the sensestrand has a 2′-deoxy modification, and both the 5′- and 3′-ends of thesense strand have a terminal cap moiety; (e) each pyrimidine nucleotideof the antisense strand has a 2′-fluoro modification, each purinenucleotide of the antisense strand has a 2′-deoxy modification, and theantisense strand has a phosphorothioate internucleotide linkage at the3′-end; and, (f) wherein the siNA molecule mediates RNA interference. 2.The siNA molecule of claim 1, wherein the terminal cap moiety is aninverted deoxy abasic moiety.
 3. The siNA molecule of claim 1, whereinsaid nucleic acid molecule includes two or more phosphorothioateinternucleotide linkages.
 4. The siNA molecule of claim 1, wherein the5′-end of the antisense strand includes a terminal phosphate group. 5.The siNA molecule of claim 1, wherein the sense strand, the antisensestrand, or both the sense strand and the antisense strand include a3′-overhang.
 6. A composition comprising the siNA molecule of claim 1,in a pharmaceutically acceptable carrier or diluent.
 7. The siNAmolecule of claim 5, wherein both the sense and the antisense strandinclude a 3′-overhang.
 8. The siNA molecule of claim 5, wherein the3′-overhang comprises 1-3 nucleotides in length.
 9. The siNA molecule ofclaim 5 wherein the overhang comprises ribonucleotides that arechemically modified at a nucleic acid sugar, base, or backbone.